姜黄素对类风湿关节炎外周血破骨细胞分化成熟及RANK基因表达的影响

发布时间：2018-03-02 00:09

  本文关键词： 类风湿关节炎 骨破坏 外周血单个核细胞 破骨细胞 姜黄素 核因子κB受体活化因子　出处：《安徽中医药大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:掌握外周血单个核细胞(PBMCs)体外诱导培养破骨细胞的方法,以此研究类风湿关节炎(RA)患者外周血破骨细胞前体细胞(OPC)的分化能力;并进一步研究姜黄素对RA患者外周血破骨细胞分化成熟及核因子κB受体活化因子(RANK)表达的影响;探讨破骨细胞在RA骨质破坏中的作用及姜黄素防治RA可能的分子机制,为中药单体姜黄素治疗RA提供新的实验依据。方法:(1)①将12例RA患者和10名健康志愿者分为RA组和健康对照组,分别抽取两组人员的外周血,分离PBMCs,采用细胞因子RANKL(100 nmol/L)和M-CSF(50 nmol/L)体外诱导培养破骨细胞的方法对细胞进行培养;对培养14天后的细胞行抗酒石酸酸性磷酸酶(TRAP)染色,观察其细胞形态;将TRAP染色阳性、胞核≥3个的细胞定义为破骨细胞并计数;对培养21天的细胞进行骨吸收实验;将RA组和健康对照组的破骨细胞形态、数量及骨吸收功能进行比较。②对RA组患者骨质破坏的临床指标进行评估,评估内容包括:X线检查其双手关节行影像学Sharp评分,双能X线骨密度仪测试其骨密度(BMD)值;并将RA组破骨细胞数量与该组骨质破坏的临床指标进行相关分析。(2)①采集3例健康志愿者PBMCs,在含不同浓度(0、2.5、5、10、20、40μmol/L)的姜黄素培养液中分别培养24、48和72 h,采用CCK-8法检测其细胞活力。②采集10例RA患者PBMCs,加入RANKL(100 nmol/L)和M-CSF(50 nmol/L)诱导PBMCs向破骨细胞分化,在细胞培养过程中加入不同浓度姜黄素进行干预;根据姜黄素浓度将实验分为4组,即空白对照组、姜黄素2.5μmol/L组、姜黄素5μmol/L组和姜黄素10μmol/L组;对培养14天后的细胞行TRAP染色,计数TRAP染色阳性、胞核≥3个的细胞(定义为破骨细胞),比较各组破骨细胞数量。③采集5例RA患者PBMCs,加入RANKL(100 nmol/L)和M-CSF(50 nmol/L)体外诱导培养,在培养过程中加入不同浓度姜黄素进行干预;实验分组同上;在细胞培养10天后,采用RT-PCR法检测各组破骨细胞前体细胞RANK m RNA的表达;Western-blot法检测各组破骨细胞前体细胞RANK蛋白的表达。结果:(1)①RA组和健康对照组的破骨细胞形态相似,无明显区别;RA组破骨细胞数量(129±6.999个/10个视野)较健康对照组(79±3.887个/10个视野)显著升高(P0.05);RA组破骨细胞骨吸收功能明显高于健康对照组。②RA组破骨细胞数量与Sharp评分呈显著正相关(r=0.810,P=0.001),与BMD(T值)呈显著负相关(r=-0.685,P=0.014)。(2)①CCK-8法检测结果显示较低浓度(2.5μmol/L、5μmol/L、10μmol/L)姜黄素对PBMCs细胞活力无明显影响,较高浓度姜黄素(20μmol/L和40μmol/L)显著降低其细胞活力。②细胞培养14天后,与空白对照组(126.3±4.1个/10个视野)比较,姜黄素2.5μmol/L组(101.8±3.5个/10个视野)、姜黄素5μmol/L组(79.9±3.8个/10个视野)、姜黄素10μmol/L组(60.6±4.4个/10个视野)破骨细胞的数量均明显减少,且具有浓度依赖性(P0.05)。③RT-PCR结果显示,与空白对照组(1.44±0.16)比较,姜黄素2.5μmol/L组(1.03±0.12)、姜黄素5μmol/L组(0.48±0.10)和姜黄素10μmol/L组(0.26±0.04)RANK m RNA的表达均显著降低,且具有浓度依赖性(P0.05);Western-blot结果显示,与空白对照组(0.68±0.11)比较,姜黄素2.5μmol/L组(0.46±0.09)、姜黄素5μmol/L组(0.36±0.08)和姜黄素10μmol/L组(0.25±0.07)RANK蛋白的表达均显著降低,且具有浓度依赖性(P0.05)。结论:(1)RA患者PBMCs向破骨细胞分化的能力明显增加,且与RA骨质破坏的临床指标密切相关。这可能是造成RA骨质破坏的重要机制。(2)姜黄素具有体外抑制RA患者PBMCs向破骨细胞分化的作用,抑制RA患者外周血破骨细胞前体细胞RANK m RNA及蛋白表达的作用,且均呈浓度依赖性。提示姜黄素可能通过作用于RANK介导的信号通路抑制了破骨细胞的分化、成熟。这可能是姜黄素治疗RA骨质破坏的重要作用机制。
[Abstract]:Objective: To investigate the peripheral blood mononuclear cells (PBMCs) induced culture of osteoclasts in vitro, in order to study the rheumatoid arthritis (RA) in peripheral blood of patients with osteoclast precursor cell (OPC) differentiation ability; and further study the effect of curcumin on RA in peripheral blood of patients with broken bone cell differentiation and nuclear receptor factor kappa B activation factor (RANK) on the expression of; to explore the molecular mechanism of osteoclasts in bone destruction in RA and the effect of curcumin in preventing RA possible, provide new experimental basis for traditional Chinese medicine monomer of curcumin in the treatment of RA. Methods: (1) the 12 cases of RA patients and 10 healthy volunteers were divided into RA group and the healthy control group, peripheral blood, extracted from the two groups using cell separation PBMCs, factor RANKL (100 nmol/L) and M-CSF (50 nmol/L) inducing culture methods on osteoclast cells were cultured in vitro; the cells cultured for 14 days for tartrate resistant acid phosphate The enzyme (TRAP) staining, observe the cell morphology; TRAP positive staining, nuclei of not less than 3 of the cells defined as osteoclasts on bone resorption and counting; experiments were performed 21 days of cell culture; the cell morphology of osteoclasts in RA group and healthy control group, and the number of bone resorption were compared. The evaluation of the clinical indicators of RA patients bone destruction, including: X-ray imaging for the joints of the hands of Sharp score, dual energy X-ray absorptiometry to test their bone mineral density (BMD) value; and the RA group was breaking clinical index of bone cell number and destruction of the group of bone were analyzed. (2) the acquisition of 3 healthy volunteers in PBMCs, with different concentrations of curcumin (0,2.5,5,10,20,40 mol/L) in the culture medium were cultured in 24,48 and 72 h, detection of cell viability by CCK-8 assay. The collection of 10 cases of RA patients with PBMCs, RANKL (100 nmol/L) and M-CSF (50 nmol/L) to induce PBMCs The differentiation of osteoclasts, with different concentrations of curcumin in cell culture in the process of intervention; according to the concentration of curcumin were divided into 4 groups, namely control group, curcumin 2.5 mol/L curcumin group, 5 mol/L group and 10 mol/L curcumin group; staining after 14 days of culture cell line TRAP, count of TRAP positive the nucleus, less than 3 of the cells (defined as osteoclasts), compared the number of osteoclasts. The collection of 5 cases of RA patients with PBMCs, RANKL (100 nmol/L) and M-CSF (50 nmol/L) cultured in vitro, with different concentrations of curcumin in the training process of intervention group are the same as above; training; 10 days after the cells were detected the expression of RANK m RNA cells broken bone cells by RT-PCR method; Western-blot method to detect the expression of RANK protein in the cell body of broken bone cells. Results: (1) RA group and healthy control group of osteoclasts morphologically similar, no 鏄庢樉鍖哄埆;RA缁勭牬楠ㄧ粏鑳炴暟閲，

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